Molecular Cloning and Sequence Analysis of FullLength cDNA Encoding Human Bone Morphogenetic Protein—7

Objective:To clone the full-length human bane morphogenetic protein-7 (BMP-7 ) gene and analyse its sequence, to aid in investigation of its function and structure. Methods : Total RNA was isolated from Chinese fetal kidney by the acid gmnidinium thiocyanate phenol-chloroform method. Two overlapping segments of human BMP- 1 cDNA were obtained by reverse transcription (RT)-PCR. Following application, the two segments were ligated to each other and subcloned into POEM-T easy vector to form PEGM-T easy/hBMP-7 recombinant plasmid. Sanger dideoxy chain-termination method was used to sequence the cDNA. Results. There was 750 bp fragment obtained RT-PCR using #2 primer from 5' end of BMP-7 gene (PCR by using # 2 and # 1) ,and 540 bp fragment from 3' end was generated by KT-PCR using # 4 primer (PCR using # 3 and # 4). Full-length cDNA encoding BMP-7 was obtained by religation of two segments. When compared with hBMP-7 sequence in Gene bank (XM30619) ,our full-length BMP-7 cDNA has a G instead of a T at nucleotide 862. This change results in valine substituting for phenylalanine in the protein. Conclusion. This is the first time that BMP-7 cDNA was successfully cloned from Chinese fetal kidney. BMP-7 cDNA plays an important role in healing injuries of the osteo-articular system. This makes BMP-7 is an attractive target far various clinical applications.

Cloning and Prokaryotic Expression of VP1 Gene of Foot-and-Mouth Disease Virus (FMDV) Type O

According to the complete genome of foot-and-mouth disease virus(FMDV)type O,a pair of special primers was designed to amplify VP1 gene.The VP1 gene was amplified by RT-PCR and subsequently inserted into the expression vector pGEX-6p-1 and induced by IPTG.Then SDS-PAGE showed the expressed protein was 51 kD in molecular weight.Then the product was purified by GSTrap FF columns.The product was detected through Western-blot that showed the protein has antigenicity.It provided fundamental data and materials for further investigation on diagnosis method of FMDV.

Cloning and Expression of the vp39 Gene of Bombyx mori Nuclear Polyhedrosis Virus in E.coli

The nuclear capsid protein gene (vp39) ofBombyx mori nuclear polyhedrosis virus (BmNPV) was amplified successfully by PCR technique and inserted into pGEM 3zf(+). The 5′ and 3′ terminal area of the amplified vp39 gene were sequenced with silver-staining dideoxy method. Bmvp39 gene was sub-cloned into the expression vector pRSET-A, and transformed intoE. coli BL21. This gene was highly expressed by IPTG induction. SDS-PAGE analysis showed that the expressed protein is about 38 kd, and the expressed amount reached maxium in 4 h with IPTG induction.

Cloning and Sequence Analysis of Carbonic Anhydrase-related Protein 10-like in Apis mellifera

[ Objective ] This study aimed to clone and analyze the gene sequence encoding carbonic anhydrase-related protein lO-like （ CARP X） from Apis mellif era. [Method] The cDNA sequence of CARPX gene was cloned through RT-PCR, and then analyzed with bioinformatic method. [Result] The full-length cDNA sequence of CARPX was 972 bp long and encoded 324 amino acid residues, including a signal peptide and two transmembrane domains. The predicted molecular mass was 37.1 kDa and the predicted isoeleetric point was 7.458. The CARP X from A. mellifera shared close relationship with proteins from Apisflorae, Bombtas impatiens, Bombus terrestris, Nasonia vitripennis and Acyrthosiphon pisum. The insect CARP X family may include two subfamilies. [ Conclusion] The results pro- vide basis for studying CARPs family.

Cloning and Sequence Analysis of IGFBP-7 Gene in Sheep

In this study, full-length CDS sequence of IGFBP-7 gene was cloned from Liangshan semi-fine wool sheep with RT-PCR method and analyzed with bioinformatics methods. The results showed that the full-length CDS sequence of IGFBP-7 gene in Liangshan semi-fine wool sheep was 846 bp in length, encoding 282 amino acids. The CDS sequence shared 99%, 95% and 90% homology with bovine, human and rat, respectively; the amino acid sequence shared 98%, 93% and 89%, respectively. The GenBank accession number was FJ589640.1. The amino acid molecular weight of IGFBP-7 was 29.0 kD, and the theoretical isoelec- tric point （pl） was 8.25. The result of phylogenetic analysis showed that IGFBP-7 gone in Liangshan semi-fine wool sheep exhibited close phylogenetic relationships with bovine, goat and other mammals, and distant phylogenetic relationships with Danio rerio and Haliotis diversicolor. IGFBP-7 gene had uniformly distributed hy- drophobic and hydrophilic regions, harboring one signal peptide, two transmembrane regions, 16 phosphorylation sites, four N-glycosylation sites and one O-glyco- sylation site. The result of secondary structure analysis showed that the random coil, or-helix and β-sheet regions accounted for 64.89%, 19.86% and 15.25%, respectively. The result of tertiary structure analysis showed that IGFBP-7 harbors an IGFBP_N domain and an Ig-like domain. This study provided scientific basis for further investigating the function of IGFBP-7 gene in sheep.

辽宁沈阳株猫泛白细胞减少症病毒VP2基因扩增及生物信息学分析

【目的】通过生物信息学方法分析1株2022年辽宁沈阳地区猫泛白细胞减少症病毒(Feline panleukopenia virus, FPV)LN3株VP2蛋白的分子特征。【方法】利用FPV胶体金试纸条对临床上表现呕吐、腹泻等症状的患病猫粪便进行检测,提取该病猫粪便的病毒DNA进行FPV VP2基因PCR扩增及测序,使用Seqman软件对序列进行拼接。将所获序列与NCBI数据库中FPV和犬细小病毒(Canine parvovirus, CPV)的VP2基因进行相似性比对及遗传进化分析。使用生物信息学软件对该毒株VP2蛋白进行预测,包括理化性质、亲/疏水性、跨膜区、糖基化位点、亚细胞定位、二级结构、三级结构等。【结果】FPV LN3株VP2基因全长1 755 bp,编码584个氨基酸;与GenBank上登录的15株FPV同属一个大分支,相似性为98.9%~99.7%;与CPV处于不同分支上。生物信息学软件预测显示,FPV LN3株VP2蛋白为亲水性蛋白,无跨膜结构;含有7个潜在N-糖基化位点、86个O-糖基化位点和50个磷酸化位点;VP2蛋白在细胞质、细胞核、线粒体中的可能性分别为43.5%、34.8%和21.7%;VP2蛋白二级结构中无规则卷曲、α-螺旋、延伸链、β-转角分别占61.82%、8.90%、24.32%及4.97%,三级结构预测结果与其一致;VP2蛋白共有20个抗原表位。【结论】VP2是FPV遗传变异的关键基因,FPV LN3株VP2蛋白属于亲水性、非跨膜稳定蛋白,共存在20个抗原表位。试验结果为进一步研究FPV VP2蛋白功能及研发新型疫苗提供理论依据。

人B组轮状病毒vp7基因的克隆和序列分析

利用逆转录-聚合酶链反应(RT-PCR)技术,扩增了人B组轮状病毒WH-2vp7基因,连接到克隆载体pUCm-T,并对其基因序列进行了分析。WH-2与ADRV的同源性达98％,与印度加尔各达分离株CAL-1达92％,而与动物B组轮状病毒同源性差别较大,如与IDIR(鼠)同源性仅为58％,与WD653(牛)B组轮状病毒同源性为63％,与ATI(牛)同源性为61％。对vp7基因的二级结构分析发现其mRNA折叠形成多达18个发卡环状结构。VP7蛋白是249个氨基酸组成,分子量为28.4kDa,含有3个潜在的N连接的糖基化位点和多个磷酰化位点,从氨基酸序列的同源性来看,WH-2与ADRV的同源性达99％,与CAL-1达95％,而IDIR仅为51％,说明了WH-2和ADRV的起源相同。此研究对了解B组轮状病毒基因的进化和变异规律具有重要意义,也将为B组轮状病毒预防性疫苗的研制提供科学的依据。

蓝舌病病毒Z1株VP7基因的克隆及序列分析

用RT PCR扩增我国蓝舌病毒Z1株的 VP7基因 ,直接将其克隆至 pGEM T载体中 ,用限制性内切酶EcoRⅠ分析经蓝 /白斑筛选和PCR鉴定的重组质粒 ,用DIG标记克隆片段制成探针与病毒基因组进行Northernblot杂交 ,证明插入片段为BTVVP 7基因特异性片段。采用Sanger双脱氧终止法测定cDNA片段的核苷酸序列 ,将这一序列与国外已发表的 9株蓝舌病毒及相关的环状病毒的VP 7基因进行了比较分析 ,结果表明 :国内BTVZ1株的VP 7基因的核苷酸序列与上述 9个毒株的同源性在 71 4%～ 98 7%之间 ,与USABTV 10株的同源性最高 ,与AUSBTV 15株的同源性最低。该研究对于我国蓝舌病基因工程疫苗的研制及对该病分子流行病学的调查有着重要意义。

蓝舌病病毒VP7基因的原核表达及反应原性分析

为获得蓝舌病病毒(BTV)VP7基因原核表达蛋白并检测其生物学活性,根据Genbank登录的BTV VP7基因,设计1对引物,运用RT-PCR扩增技术获得BTV VP7目的片段,然后定向克隆到pPROEXHTb原核表达载体上,再转化重组质粒pPROEXHTb-VP7到BL21感受态细胞,IPTG诱导,表达产物纯化,进行SDS-PAGE鉴定及Western-blot反应原性分析.结果表明:表达的VP7目的蛋白大小为35ku,Western-blot分析表明纯化后的蛋白可以与BTV单克隆抗体发生特异性反应.

Rice bicoid-related cDNA sequence and its expression during early embryogenesis

Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation. To clone and characterize the rice bicoid-related genes, one cDNA clone, Rb24 (EMBL accession number: AJ2771380), was isolated by screening of rice unmature seed cDNA library. Sequence analysis indicates that Rb24 contains a putative amino acid sequence, which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity, 75% similarity) and involves a lys-9 in putative helix 3. Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner. The transcript was detected strongly in young panicles, but less in young leaves and roots. This results are further confirmed with paraffin section in situ hybridization. The signal is intensive in rice globular embryo and located at the apical tip of the embryo, then, along with the development of embryo, the signal is getting reduced and transfers into both sides of embryo. The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

Polymorphic Analysis of TLR4 Gene in Hainan Local Pig Breeds Based on Sequencing Technology

[Objective]The paper was to understand the polymorphism of TLR4 gene in Hainan local pig breeds.[Method]The TLR4 genome sequence of Wuzhishan pigs,Lingao pigs,and Tunchang pigs was cloned and sequenced by PCR.The sequence was analyzed using DNAStar and BioEdit software,and the structure of TLR4 protein was analyzed using SMART,SOPMA,and SWISS-MODEL online software.The secondary structure and tertiary structure of TLR4 protein were predicted as well.[Result]The total length of TLR4 gene in three Hainan local pig breeds were all 10435 bp which included a 2526 bp CDS(coding 841 amino acid).Intra-specific comparison showed that there was one nucleotide site with polymorphism in Wuzhishan pigs;there were six nucleotide sites with polymorphism in Tunchang pigs,two of which were located in the coding area;there were three nucleotide sites with polymorphisms in Lingao pigs,one of which was located in the coding area.When interspecific comparisons of TLR4 gene sequences from three Hainan local pig breeds were performed,there were 27 nucleotide sites with polymorphism,two of which were missense mutations,resulting in amino acid changes.The homology of TLR4 gene sequence from three pig breeds was 99.8%-99.9%,indicating that the TLR4 gene sequence was highly conservative.The predicted protein structure indicated that the G→T mutation at 7209 site and G→A mutation at 7781 site in TLR4 gene of Hainan local pig breeds caused changes in the secondary and tertiary structure of TLR4 protein.[Conclusion]TLR4 gene in Hainan local pig breeds has polymorphism.The structural changes caused by polymorphism may change the function of TLR4 gene.

鸡MDA5的序列分析与结构域预测

为了解鸡黑色素瘤分化相关基因-5(Melanoma differentiation-associated protein 5,MDA5)的序列和结构域,试验使用PCR方法扩增了鸡MDA5基因并构建真核表达质粒,实现了鸡MDA5蛋白的真核表达,同时利用MegAlign等软件对鸡MDA5氨基酸序列进行分析。结果显示:鸡MDA5全长3 006 bp,编码1 001个氨基酸;鸡MDA5与人、猕猴、野猪、小鼠、绿头鸭、灰雁、鸿雁、鲫鱼、草鱼的氨基酸相似性分别为62.4%、62.9%、62.7%、64.1%、86.8%、87.3%、87.4%、48.1%、46.3%;鸡MDA5与鸟类进化关系较近,与哺乳动物次之,与鱼类进化关系较远;鸡MDA5由N端两个半胱天冬酶激活招募结构域、DExD/H box RNA解旋酶结构域和C端结构域组成;鸡MDA5二级结构包含α螺旋(50.95%)、延伸链(11.19%)、β转角(3.9%)和无规则卷曲(33.97%),三级结构与人MDA5类似,在空间上呈现半闭合环状结构。研究成功鉴定了鸡MDA5基因,分析了其基因序列并预测其结构域,为深入研究鸡MDA5结构与功能及其在鸡抗病毒天然免疫应答中的作用奠定基础。

水貂AANAT基因克隆及生物信息学分析

【目的】试验旨在对美洲水貂(Neovison vison)褪黑素(N-acetyl-5-methoxytryptamine,MT)合成的限速酶5-羟色胺-N-乙酰基转移酶(aralkylamine N-acetyltransferase,AANAT)基因进行克隆和生物信息学分析,为探究AANAT基因在水貂中的生物学功能提供参考。【方法】对水貂尾静脉采血后提取DNA,利用同源重组的方法克隆AANAT基因并分析其编码区(CDS)序列,推导成氨基酸序列后进行相似性比对及系统进化树构建,并对AANAT蛋白进行生物信息学分析。【结果】水貂AANAT基因序列长约1631 bp,CDS区长504 bp,可编码167个氨基酸。水貂AANAT氨基酸序列与雪貂相似性最高(98.2%),系统进化树分析也显示其与雪貂亲缘关系最近。生物信息学分析结果表明,水貂AANAT蛋白为疏水性蛋白,无跨膜结构域和信号肽,主要分布于细胞质中,有5个抗原结合位点为非分泌型蛋白,该蛋白含有多个磷酸化、糖基化等翻译后修饰位点。AANAT蛋白含有1个保守的N-酰基转移酶超家族结构域,该家族与哺乳动物的昼夜节律密切相关。AANAT蛋白二级结构以无规则卷曲为主,三级结构含有多个保守的催化残基。蛋白互作网络分析表明,AANAT蛋白可与多个5-羟色胺合成相关的蛋白相互作用。【结论】试验成功获得水貂AANAT基因序列,AANAT蛋白结构和特异性修饰位点可能通过定位乙酰辅酶A和5-羟色胺参与调控MT的生物合成,结果可为AANAT基因对水貂胚胎滞育的调控机制及提高繁殖力等研究提供参考。

水稻胞质核糖体蛋白基因OsRPS7的克隆与序列分析

以已公布的黑麦胞质核糖体蛋白基因ScRPS7的cDNA序列为信息探针,在中国华大水稻基因组数据库中搜索与之高度同源的基因组重叠群。采用计算机拼接和RT PCR方法克隆了水稻胞质核糖体蛋白基因的全长cD NA序列,命名为OsRPS7。该cDNA序列全长919bp,编码192个氨基酸;其与黑麦、拟南芥和芸薹的S7核糖体蛋白的氨基酸一致率分别为88%、72%和72%。对OsRPS7的基因组结构和基因的功能进行了分析和预测。

家蚕丝氨酸蛋白酶基因BmHP21的克隆及表达分析

丝氨酸蛋白酶参与昆虫对抗入侵病原体及保护受伤组织的黑化反应。为了探讨家蚕黑化反应过程中丝氨酸蛋白酶在酚氧化酶原前体级联体系的作用,克隆了家蚕的一个新的丝氨酸蛋白酶基因,命名为BmHP21(GenBank登录号:JF431073)。该基因由1 233个核苷酸组成,编码410个氨基酸。通过SMART网站预测蛋白结构显示BmHP21包括一个clip结构域和一个胰蛋白酶结构域。BmHP21蛋白和烟草天蛾Ms-HP21的氨基酸序列有55%的相似性,推测BmHP21在家蚕黑化反应中起着激活酚氧化酶原前体激酶(PPAE)的作用。RT-PCR检测结果表明,BmHP21在家蚕大部分组织包括脂肪体中都有表达,其mRNA转录几乎贯穿于整个家蚕的幼虫、蛹和成虫发育阶段,推测BmHP21除了参与黑化反应外,还有其它的生理功能。

家蚕模式识别受体PGRP、βGRP编码基因的克隆鉴定及表达谱分析

模式识别受体(PRR)是生物体先天免疫系统中免疫受体的代表,对生物体的生存极为重要。克隆鉴定了家蚕的14个模式识别受体编码基因,包括10个肽聚糖识别蛋白(PGRP)基因和4个β-葡聚糖识别蛋白(βGRP)基因,并得到了BmPGRP-S3、BmPGRP-S4和BmPGRP-L5的完整编码序列。家蚕PGRP的长型和短型亚家族都具有典型的酰胺酶活性结构域,短型亚家族具有信号肽,长型亚家族则没有信号肽。5个PGRP长型亚家族基因成簇分布于第1号染色体;5个PGRP短型亚家族基因中有2个分布于第9号染色体,有3个分布于第16号染色体。家蚕βGRP家族成员都具有信号肽,其中BmβGRP1-3成簇分布于第11号染色体,编码蛋白不具有典型的葡聚糖结合结构域;BmβGRP4独立分布于第22号染色体,编码蛋白具有典型的葡聚糖结合结构域。基因芯片数据分析表明,BmPGRP-L5和BmβGRP1在5龄第3天幼虫各组织中没有表达,其余12个模式识别受体基因为多组织表达,但在丝腺组织中均无表达。在这12个模式识别受体基因中,BmPGRP-L3等6个模式识别受体基因在中肠组织中的表达水平偏高;BmβGRP3、BmβGRP4和BmPGRP-L3、BmPGRP-L4等在家蚕生殖腺中的表达水平较高。在生殖腺以外的其他各组织中,这12个基因的表达不具有雌雄差异性。BmβGRP1在家蚕各发育时期没有表达,BmPGRP-L5主要在变态发育的转折期表达,其余12个模式识别受体基因在各发育时期均有表达,并从5龄第3天幼虫到上蔟第2天有较高水平的表达,雌雄个体间无表达差异性。由此说明这些模式识别受体基因的表达具有一定的组织性和发育时期性。给人工饲料无菌饲养的家蚕5龄第3天幼虫分别添食大肠杆菌、家蚕黑胸败血菌和家蚕白僵菌,对免疫诱导3、6、12和24h的家蚕进行个体水平的实时荧光定量PCR检测,发现PGRP长型亚家族基因BmPGRP-L1、BmPGRP-L3和短型亚家族基因BmPGRP-S1-3均能被这3种微生物诱导上调表达;βGRP基因家族中的BmβGRP3、BmβGRP4也能被3种微生物诱导上调表达。同时对诱导12h时家蚕各组织中14个家蚕模式识别受体基因的表达谱进行分析,结果显示经3种微生物分别诱导后,家蚕头部组织中BmβGRP2、BmβGRP4、BmPGRP-L2和BmPGRP-S1、BmPGRP-S3-5均上调表达;表皮、中肠和脂肪体中仅有BmβGRP3、BmPGRP-L4等少数模式识别受体基因上调表达。

番鸭细小病毒国内分离株主要结构蛋白基因的克隆和序列分析

根据国外已发表的番鸭细小病毒 (MPV) FM株基因组核苷酸序列 ,设计了 1对引物 ,分别对国内 2株番鸭细小病毒分离株 MPV扬州 (YZ)株和 MPV佛山 (FS)株主要结构蛋白 (VP2和 VP3)基因进行 PCR扩增 ,并克隆到p CR3.1T载体 ,经酶切鉴定筛选出阳性克隆质粒并进行核苷酸序列分析比较。结果表明 ,MPV YZ和 MPV FS的VP2 - VP3基因大小均为 176 4bp,编码 5 88个氨基酸。 MPV YZ、MPV FS与 MPV FM核苷酸序列同源性分别为98.5 %和 98.6 % ,氨基酸序列同源性为 97.1%和 97.8% ;MPV YZ与 MPV FS核苷酸序列的同源性为 99.5 % ,氨基酸序列同源性为 98.8%。

重组质粒pEGFP-BMP7的构建及在大鼠骨髓间充质干细胞中的表达

目的体外构建重组质粒pEGFP_BMP7,并检测其在大鼠骨髓间充质干细胞中的表达。方法应用RT_PCR方法从大鼠肾脏中分离、扩增目的基因片段,并进行测序,随后将所得cDNA定向克隆到真核表达载体pEGFP_N1中,进行双酶切来鉴定克隆的正确性。通过脂质体介导重组pEGFP_BMP7瞬时转染大鼠骨髓间充质干细胞,确定转染效率,并通过RT_PCR、免疫细胞化学手段检测BMP7的表达。结果通过RT_PCR成功获得1.3 kb的cDNA片段,该cDNA除756 bp处有一碱基从T突变成A外,其余序列与大鼠BMP7基因完全相符。重组质粒双酶切图谱显示,BMP7 cDNA被正确插入载体中。绿色荧光蛋白在大鼠骨髓间充质干细胞中早期瞬时转染效率可达33%。转染后RT_PCR和免疫细胞化学检测证实重组pEGFP_BMP7在大鼠骨髓间充质干细胞中的表达。结论成功构建重组真核表达质粒pEGFP_BMP7,并在大鼠骨髓间充质干细胞中得到表达,有助于应用BMP7行基因治疗,促进牵张成骨、骨痂形成和修复颅颌面骨缺损。

猪细小病毒非结构蛋白NS1基因的克隆、序列分析及蛋白质结构预测

根据GenBank登录的猪细小病毒NADL-2株和China株序列,利用Oligo 6.0软件设计1对扩增NS1全基因的特异性引物,对从贵州省安顺地区检测到的PPV的NS1基因进行扩增、克隆、测序、序列分析和蛋白质结构预测分析。测序结果表明,扩增的目的基因长度为1986bp,共编码659个氨基酸,其中NS1基因的1287、1288和1298位碱基发生了缺失,导致429、430和433位氨基酸发生缺失。系统发生树结果表明,本试验测序的NS1基因与NADL-2弱毒株处在同一进化分支;氨基酸同源性与NADL-2弱毒株和Kresse毒株最高,均为98.6%。蛋白质结构预测分析结果表明,非结构蛋白NS1的分子质量为75269.76u,理论等电点pI为7.25,不稳定系数为41.57,推测其为不稳定蛋白质;脂肪指数为73.58,总体平均亲水性为-0.565,推测该蛋白质是一种亲水性蛋白质;该蛋白质含有丰富的α-螺旋、β-折叠、β-转角和无规则卷曲,柔性区域较多,呈连续分布;非结构蛋白NS1无跨膜区和信号肽;预测非结构蛋白NS1含有3个N-糖基化位点、3个cAMP和cGMP依赖性蛋白激酶磷酸化位点、5个蛋白激酶C磷酸化位点、22个酪蛋白激酶Ⅱ磷酸化位点、1个酪氨酸激酶磷酸化位点、8个N-肉豆蔻酰化位点、1个酰胺化位点及1个ATP/GTP结合位点基序A(P-环);该蛋白质抗原表位较多,存在10个主要的抗原表位。

松墨天牛OBP基因MaltOBP2和MaltOBP6的克隆、序列分析及组织表达谱和原核表达研究

【目的】本研究旨在探索松墨天牛Monochamus alternatus Hope在嗅觉识别寄主植物过程中扮演重要角色的气味结合蛋白(odorant binding proteins,OBPs)的结构及功能。【方法】利用生物信息学方法对得到的Malt OBP2和Malt OBP6基因序列和蛋白结构进行分析,并通过实时荧光定量PCR分析Malt OBP2和Malt OBP6在松墨天牛雄虫不同组织和时空中的表达差异,利用p ET32a(+)原核表达载体对Malt OBP2和Malt OBP6进行了诱导蛋白表达。【结果】本研究得到两个松墨天牛气味结合蛋白基因——Malt OBP2(Gen Bank登录号:KP120891)和Malt OBP6(Gen Bank登录号:KP120892),ORF长度分别为402 bp和408 bp,翻译的氨基酸序列均含有4个保守的半胱氨酸位点,表明得到的两个OBP基因的编码蛋白均属于Minus-C OBP亚家族;推导的两个OBP蛋白均有6个α螺旋区域,且α螺旋区域在两个蛋白的位置非常相似,但是两个OBP蛋白推测的配体结合位点和位点极性却完全不同。组织表达模式表明,Malt OBP2和Malt OBP6在成虫头部、触角、下颚(唇)须、腹部末端和足中均有表达,表达程度不一,但都在头部显著表达,触角中的表达量相比其他组织中较低或只是持平。发育表达结果表明,Malt OBP2在蛹触角中的表达量最高,而Malt OBP6在幼虫头部的表达量最高。本研究成功构建了原核表达载体p ET32aMalt OBP2和p ET32a-Malt OBP6,并进行了OBP蛋白诱导表达,低温(16℃和20℃)条件利于蛋白表达在上清液中,延长诱导表达时间(12 h)可以增加蛋白的表达量。【结论】本研究从松墨天牛体内得到了Minus-C OBP蛋白亚家族的两个基因Malt OBP2和Malt OBP6,通过配体结合位点推测它们具有不同的生理功能;通过组织表达谱结果推测这两个OBP基因在松墨天牛中的功能不仅仅局限于嗅觉识别,或还有味觉感受、化学感受等其他生理功能。本研究结果为两个OBP蛋白的结构和功能研究奠定了基础,为探索松墨天牛的化学感受机制提供了条件。